HAEMOPOIETIC MATURATION FACTOR

Abstract: Disclosed is a human maturation factor polypeptide and DNA(RNA) encoding such haemopoietic maturation factor polypeptides. Also provided is a procedure for producing such polypeptide by recombinant techniques and antibodies against such polypeptide. Such polypeptides may be combined with a suitable pharmaceutical carrier or diluent to provide diagnostic, therapeutic and/or prophylactic effects against various diseases related to the underexpression of such human haemopoietic maturation factor polypeptide.

Description: HAEMOPOIETIC MATURATION FACTOR This invention relates to newly identified polynucleotide sequences, polypeptides encoded by such sequences, the use of such polynucleotideε and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is a haemopoietic factor. Various growth factors have been discovered, studied and utilized (cellular and molecular biology, edited by the Japanese Tissue Culture Association, Asakura Shoten (1987). Such cell growth factors include epidermal growth factor, platelet derived growth factor, acidic fibroblast growth factor and basic fibroblast growth factor. All these factors have been isolated based upon growth promotion of fibroblast cells. However, these factors have also been found to display widely ranging activity and poor specificity. Accordingly, recent attempts have been made to search for growth factors specifically acting on functionally differentiated cells. As a result, growth factors such as keratinocyte growth factor and hepatocyte growth factor have been isolated, thus creating the possibility that these factors could be used to treat diseases vulnerable to -1- SUBSftTUTE SHEET (RULE 26) their specific action spectra. Another growth factor which has been isolated is disclosed in European Patent Application No. 92102385.9 applied for by Takeda Chemical Industries, Ltd. disclosing a glia activating factor which has glial cell growth promoting activity, and the DNA encoding for that polypeptide. Haematopoiesis is the production of blood cells. The major haematopoietic tissues are bone marrow, spleen, lymph nodes and thy uε. Haematopoiesis in the human embryo begins in the second week of life. Bone marrow appears in the embryo in the second month, and it becomes the dominant haematopoietic organ in the latter half of gestation and throughout postnatal life. The bone marrow contains stem cells that give rise to all cells of the haemopoietic series. All of the blood cells except T-lymphocytes are produced in the marrow. Haematopoietic stems cells differentiate into the mature blood cells in response to the action of haemopoietic maturation factor. Accordingly, a haematopoietic growth factor which could be purified and isolated, would have extensive therapeutic value in the treatment and diagnosis of blood cell and other diseases. In accordance with one aspect of the present invention, there is provided a novel polypeptide which is a haemopoietic maturation factor, as well as analogs and derivatives thereof. The haemopoietic maturation factor of the present invention is of human origin. In accordance with another aspect of the present invention, there is provided a polynucleotide (DNA or RNA) which encodes such polypeptide. In accordance with still another aspect of the present invention, there is provided a procedure for producing such polypeptide by recombinant techniques. In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptide, or DNA sequence encoding such polypeptide for -2- SUBSTlTUTtSHEET(RULE26) therapeutic purposes, for example stimulating differentiation and proliferation of cells of haemopoietic or neural origin. In accordance with yet another aspect of the present invention, there is provided an antibody against the haemopoietic maturation factor. In accordance with yet another aspect of the present invention, there is provided a composition which is employed as an antagonist to haemopoietic maturation factor, e.g., an antibody against such polypeptide which may be used to inhibit the action of such polypeptide, for example, in the treatment of T-cell deficient related disorders. These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein. The following drawings are meant only as illustrations of specific embodiments of the present invention and are not meant as limitations in any manner. FIG. 1 shows the polynucleotide sequence which encodes for the haemopoietic maturation factor polypeptide. It also shows the amino acid sequence of the haemopoietic maturation factor polypeptide. The standard three letter abbreviation has been used to depict the amino acid sequence. FIG. 2 shows.the homology of the haemopoietic maturation factor to glia maturation factor β. FIG. 3 illustrates the banding characteristics of the purified haemopoietic maturation factor protein after bacterial expression, and purification. FIG. 4 is a Northern Blot analysis indicating the organs in the human body in which the haemopoietic maturation factor is predominantly found. FIG. 5 illustrates that Hela cell growth is inhibited in the presence of haemopoietic maturation factor. In accordance with one aspect of the present invention, there is provided a DNA sequence (and corresponding RNA sequence) as set forth in Figure 1 of the drawings and/or DNA (RNA) sequences encoding the same polypeptide as the sequence of Figure 2 of the drawings, as well as fragment portions, derivatives, analogs and all allelic variants of such sequences. -4- SUfeTITUTfc SHEET (RULE 26) In accordance with another aspect of the present invention, there is provided a polynucleotide which encodes the same polypeptide as the polynucleotide of the cDNA clone deposited as ATCC deposit number 75514, deposited on August 4, 1993, and/or fragments, analogs, derivatives or allelic variants of such polynucleotide. In the case of DNA, DNA may be single stranded or double stranded, and if single stranded the DNA sequence may be the "sense" strand shown in Figure 1 or the one complementary thereto. The polynucleotide (DNA or RNA, preferably DNA) includes at least the portion coding for the polypeptide, which coding portion may be the same as that in the deposited clone or may be different than that in the deposited clone provided that it encodes for the same polypeptide or an allelic variant thereof. The coding portion preferably encodes at least the mature form of the protein of the present invention. The present invention further relates to polynucleotide sequences which hybridize to the hereinabove-described polynucleotide sequences if there is at least 50% and preferably at least 70% identity between the sequences. In another preferred embodiment, the present invention relates to polynucleotide sequences which hybridize under stringent conditions to the hereinabove- described polynucleotide sequences. As herein used, the term "stringent conditions" means hybridization will occur if there is at least 95% and preferably at least 97% identity between the segments. Thus, the present invention includes DNA (RNA) sequences encoding allelic variant forms of the peptide encoded by the DNA of Figure 1. Thus, the present invention provides isolated DNA (RNA) encoding for a naturally occurring human polypeptide which is a haemopoietic maturation factor, as well as allelic variants thereof. The present invention further relates to a polypeptide which is a haemopoietic maturation factor, and which has the structure shown in Figure 2, as well as allelic variants thereof, and analogs, fragments and derivatives thereof which have the same function as the naturally occurring polypeptide. The polypeptide of the present invention is mainly expressed in haemopoietic tissues and , as such, represents an haemopoietic maturation factor for controlling cells of haemopoietic origin and cells with which they interact. The present invention further relates to a polypeptide encoded by the DNA contained in the clone deposited as ATCC number 75514 on August 4, 1993 as well as analogs, fragments, derivatives and allelic variants thereof. These deposits will be maintained under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purposes of Patent Procedure. These deposits are provided merely as a convenience and are not an admission that a deposit is required under 35 U.S.C. § 112. The sequence of the polynucleotides contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with the description of sequences herein. A license may be required to make, use or sell the deposited materials, and no such license is hereby granted. The polypeptide of the present invention is preferably provided in an isolated form, and preferably is purified. The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring) . For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, separated from some or all of the coexisting materials in the natural system, -6- SU β STlTUTtSHEET(RULE26) is isolated. Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment. In a preferred embodiment, the haemopoietic maturation factor is a full length mature human haemopoietic maturation factor or an allelic or glycosylated variant thereof. The polynucleotide may also encode a preprotein which is processed and secreted from mammalian cells as the mature protein. The polynucleotide sequence of the present invention may encode for a mature form of the polypeptide or it may also encode for a leader sequence which facilitates secretion of the polypeptide. For example, the desired DNA sequence may be fused in the same reading frame to a DNA sequence which aids in secretion of the polypeptide, for example a leader sequence which functions as a secretory sequence for controlling transport of the polypeptide from the cell of the host. The protein having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to form the mature form of the protein. The polynucleotide of the present invention may also be fused in frame to a marker sequence, for example, a histidine tag, which allows for purification of the polypeptide. Thus, the polypeptide(s) of the present invention may be the mature form of the haemopoietic maturation factor of the present invention; or may be in the form of a preprotein or prepolypeptide wherein the mature polypeptide includes a leader or secretory sequence; or may be in the form of a fusion protein wherein additional amino acids which aid in, for example, purification of the polypeptide are fused to the mature or preprotein at either the 3' or 5' end thereof. -7- SUBSTϊTUTt SHEET (RULE 26) In a preferred embodiment of the present invention, the marker sequence is a hexa-hiεtidine tag added to the coding frame by a bacterial expression vector. The polypeptide of the present invention is found predominantly in the spleen and thymus and is most concentrated in the leukocytes. As hereinabove indicated, the present invention also includes variants of the polypeptide which is encoded by the DNA of Figure 1 and/or variants of the DNA contained in the deposited clone, which retains the qualitative activity of such a polypeptide which is a haemopoietic maturation factor. The variant may be a substitutional variant, or an insertion variant or a deletional variant. Such variants can be naturally occurring allelic variants such as for example, those with different glycosylation patterns or substitution at the amino acid level or deletion at the amino acid level. Such variants may also be produced by site specific mutagenesis. The substitutional variant may be a substituted conserved amino acid or a substituted non- conserved amino acid, and preferably a conserved amino acid. A polynucleotide encoding a polypeptide of the present invention may be obtained from a cDNA library derived from human early stage kidney tissue, spleen, thymus, or from leukocytes. It contains an open reading frame encoding a mature protein of 142 amino acids in length. The amino acid of the haemopoietic maturation factor has sequence homology to a known growth factor isolated from neural cells. The polypeptide encoded by the polynucleotide is structurally related to human glia maturation factor with 82% identical amino acids and 92% similarity over the entire region. It is also structurally related to bovine glia maturation factor with 82% identical amino acids and -8- sy β smυ t SHEET (R ε 26) 92% similarity over the entire coding region. The polypeptide may be found on the surface of T-cellε. Glia maturation factor is a 17 kd protein found in the brain of mammals with no apparent sequence homology to other known proteins. It is most abundant in the embryo but is also found in later stages of development. The human glial maturation factor controls the differentiation and proliferation of certain cells of neural origin. Host cells are transformed with the expression vectors of this invention and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformantε or amplifying the haemopoietic maturation factor gene. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan. "Transformation" means introducing DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integration. Unless indicated otherwise, the method used herein for transformation of the host cells is the method of Graham, F. and van der Eb, A., Virology 52:456-457 (1973). However, other methods for introducing DNA into cells such as by nuclear injection or by protoplast fusion may also be used. If prokaryotic cells or cells which contain substantial cell wall constructions are used, the preferred method of transfections is calcium treatment using calcium chloride as described by Cohen, F.N. et al . , Proc. Natl. Acad. Sci. (USA), 69:2110 (1972). "Transfection" refers to the introduction of DNA into a host cell whether or not any coding sequences are ultimately expressed. Cells do not naturally take up DNA. Thus, a variety of technical "tricks" are utilized to bypass natural barrierε to gene transfer. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, CaP0 4 and electroporation. Transformation of the host cell is the indicia of εucceεεful tranεfection. The polynucleotide of the present invention may be employed for producing a polypeptide by recombinant techniques. Thus, for example, the polynucleotide sequence may be included in any one of a variety of vectors or plasmids for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA'ε; yeaεt plasmids; vectors derived from combinations of plasmids and phage DNAs, viral DNA such as vaccinia, adenoviruε, fowl pox virus, and pseudorabies. However, any other plasmid or vector may be used as long as it is replicable and viable in the host. As hereinabove described, the appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(ε) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art. The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(ε) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp. the phage lambda PL promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression. In addition, the expression vectors preferably contain a gene to provide a phenotypic trait for selection of -10- SUBS UTE SHEET {RULE 26) transformed host cells such as dihydrofolate reductaεe or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli. The vector containing the appropriate DNA sequence as hereinabove deεcribed, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein. As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli. Salmonella typhimurium: fungal cells, such as yeast; animal cells such as CHO or Bowes melanoma; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein. More particularly, the present invention also includes recombinant conεtructε compriεing one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inεerted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter and an enhancer, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example. Bacterial: pQE-9 (Qiagen), pBs, phagescript, PsiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNHlβa, pNH46a (Stratagene) ; pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector may be used as long as it is replicable and viable in the host. -11- SUBSTITUT SHE£T (RULE 26) Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial promoterε include lad, lacZ, T3, T7, gpt, lambda P R , and trc. Eukaryotic promoters include CMV immediate early, HSV thymidine kinaεe, early and late SV40, LTRs from retroviruε, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. In a further embodiment, the present invention relates to host cells containing the above-described construct. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the hoεt cell can be a prokaryotic cell, εuch aε a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE, dextran mediated transfection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology. 1986). The constructs in host cells can be uεed in a conventional manner to produce the gene product coded by the recombinant sequence. Alternatively, the encoded polypeptide can be synthetically produced by conventional peptide synthesizers. Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free tranεlation εyεtems can also be employed to produce εuch proteinε using RNAs derived from the DNA conεtructε of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hostε are deεcribed by Sambrook, et al.. Molecular Cloning; A Laboratory Manual. Second Edition, (Cold Spring Harbor, N.Y., 1989), the disclosure of which is hereby incorporated by reference. Transcription of a DNA encoding the polypeptides of the present invention by higher eukaryotes iε increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elementε of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side of the replication origin (bp 100 to 270), a cytomegalovirus early promoter enhancer, a polyoma enhancer on the late side of the replication origin, and adenovirus enhancerε. Generally, recombinant expression vectors will include originε of replication and εelectable markers permitting transformation of the hoεt cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRPl gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operonε encoding glycolytic enzymeε εuch aε 3-phoεphoglycerate kinase (PGK), α-factor, acid phosphatase, or heat shock proteins, among others. The heterologouε structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Useful expresεion vectorε for bacterial uεe are conεtructed by inεerting a structural DNA sequence encoding a desired protein together with suitable tranεlation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli. Bacillus subtilis. Salmonella tyohimurium and various εpecieε within the genera Pεeudomonas, Streptomyces , and Staphylococcuε, although otherε may alεo be employed aε a matter of choice. Aε a repreεentative but nonlimiting example, useful expresεion vectorε for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmidε compriεing genetic elementε of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectorε include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA). These PBR322 "backbone" sectionε are combined with an appropriate promoter and the structural sequence to be expressed. Following transformation of a suitable host strain and growth of the host εtrain to an appropriate cell denεity, the selected promoter is derepreεsed by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification. Haemopoietic maturation factor is recovered and purified from recombinant cell cultures by methods used heretofore, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography (e.g., using DNA or nucleotides on a solid support), hydroxyapatite chromatography and lectin chromatography. It is preferred to have low concentrations (approximately 0.1-5mM) of calcium ion present during purification (Price, et al., J. Biol. Che .. 244:917 (1969)). -14- SUβSmUTE SHEET (RULE 26) Various mammalian cell culture εystems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblastε, deεcribed by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expreεεion vectorε will compriεe an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequenceε, and 5' flanking nontranεcribed εequenceε. DNA εequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation εiteε may be uεed to provide the required nontranεcribed genetic elements. Recombinant protein produced in bacterial culture is usually isolated by initial extraction from cell pellets, followed by one or more salting-out, aqueous ion exchange or size exclusion chromatography steps. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification stepε. Microbial cellε employed in expreεεion of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. The polypeptide of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniqueε from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cellε in culture) of a polynucleotide sequence of the present invention. Depending upon the hoεt employed in a recombinant production procedure, the polypeptideε of the present invention may be glycosylated with mammalian or other eukaryotic carbohydrates or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue (at poεition minus 1). In addition to naturally occurring allelic forms of the polypeptide, the present invention also embraces analogs and fragments thereof, which also function as a haemopoietic maturation factor. Thuε, for example, one or more of the amino acid reεidueε of the polypeptide may be replaced by conserved amino acid residues. The haemopoietic maturation factor may be used in the treatment of abnormalitieε of neural or haemopoietic origin, for example cancer, εince the haemopoietic maturation factor may block or retard the growth of cancerous cells. The polypeptide of the present invention may also be used for treating leukemia. Leukemia is clinically featured by suppreεsion of normal blood cell formation resulting from suppresεion of normal haemopoieεiε by factorε produced by leukemic cells. This results in anemia, thrombocytopenia and granulocytopenia. Accordingly, administration of a therapeutically effective amount of haemopoietic maturation factor may be used to overcome the suppression of haemopoiesis by leukemic cells. Haemopoietic maturation factor may also be used for treating other blood related disorders, e.g., hemolysiε, polycythemia vera, melogibroεiε, hemophilia, and εplenomegaly. Haemopoietic maturation factor may be used in the stimulation of differentiation of mature blood cellε in situations where a patient has undergone a bone marrow transplant due to a cancer or other situation. Haemopoietic maturation factor may also be used to stimulate bone marrow cells in vitro for research and diagnostic purposes. The polypeptide of the present invention may also toe employed in accordance with the present invention by expression of such polypeptide in vivo , which iε often referred to aε "gene therapy." Thuε, for example, cellε εuch aε bone marrow cellε may be tranεduced with a polynucleotide (DNA or RNA) encoding for the polypeptide ex vivo, the tranεduced cellε are then provided to a patient to be treated with the polypeptide. Such methodε are well-known in the art. For example, cells may be tranεduced by procedureε known in the art by uεe of a retroviral particle containing RNA encoding for the polypeptide of the present invention. Similarly, transduction of cells may be accomplished in vivo for expression of the polypeptide in vivo, for example, by procedures known in the art. As known in the art, a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the present invention may be administered to a patient for transduction in vivo and expression of the polypeptide in vivo . These and other methods for administering a polypeptide of the present invention by εuch methodε εhould be apparent to those skilled in the art from the teachingε of the present invention. For example, the expression vehicle for transducing cells may be other than a retroviral particle, for example, an adenoviruε, which may be used to transduce cellε in vivo after combination with a suitable delivery vehicle. The polypeptide of the present invention may be employed in combination with a suitable pharmaceutical carrier. Such compositions comprise a therapeutically effective amount of the protein, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but iε not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration. -17- SUBST1TUTE SHEET (RULE 26) An injection of a composition containing the haemopoietic maturation factor may be prepared by conventional methodε using, for example, physiological saline or aqueous solutionε containing glucose or other auxiliary agents. The pharmaceutical compositions such as tablets and capsuleε can also be prepared in accordance with conventional methods. The injections, solutionε, tabletε and capsules aε the pharmaceutical compoεitionε are prepared under aseptic conditions. The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositionε of the invention. Aεsociated with such container(s) can be a notice in the form preεcribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the polypeptide of the present invention may be employed in conjunction with other therapeutic compounds. When the haemopoietic maturation factor of the present invention is used as a pharmaceutical, it can be given to mammals (εuch aε humans, mice, rats, hamster, dogs, rabbits and cats), in a suitable vehicle. When the polypeptide of the present invention iε used as a pharmaceutical as deεcribed above, it iε given, for example, to the above-mentioned εubjectε in therapeutically effective doses of at least about 10 μg/kg body weight and in most cases it would be administered in an amount not in excesε of about 8 mg/kg body weight per day and preferably the doεage is from about 10 μg/kg body weight to about 1 mg/kg body weight daily, taking into account the routes of administration, symptoms, etc. Each of the cDNA sequences identified herein or a portion thereof can be used in numerous ways as polynucleotide reagents. The sequenceε can be used as diagnostic probes for the presence of a specific mRNA in a particular cell type. In addition, these sequenceε can be used as diagnostic probes suitable for use in genetic linkage analysiε (polymorphiεmε) . The εequenceε of the preεent invention are alεo valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a current need for identifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymorphiεmε) are presently available for marking chromosomal location. The mapping of cDNAs to chromosomes according to the present invention is an important first step in correlating thoεe εequenceε with geneε associated with disease. Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the cDNA iε used to rapidly select primers that do not span more than one exon in the genomic DNA, thuε complicating the amplification proceεs. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment. PCR mapping of somatic cell hybrids iε a rapid procedure for assigning a particular DNA to a particular chromosome. Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogouε manner. Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by -19- SUBSriTUTE SHEET (RULE 26) hybridization to construct chromosome specific-cDNA libraries. Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with cDNA as short as 500 or 600 baseε; however, cloneε larger than 2,000 bp have a higher likelihood of binding to a unique chromoεomal location with εufficient εignal intenεity for εimple detection. FISH requireε use of the clone from which the EST was derived, and the longer the better. For example, 2,000 bp iε good, 4,000 iε better, and more than 4,000 iε probably not necessary to get good resultε a reasonable percentage of the time. For a review of this technique, εee Verma et al.. Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988). Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. (Such data are found, for example, in V. McKuεick, Mendelian Inheritance in Man (available on line through Johns Hopkins Univerεity Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes). Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease. With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential cauεative geneε. (Thiε aεεumeε 1 megabaεe mapping reεolution and one gene per 20 kb). Co pariεon of affected and unaffected individualε generally involves first looking for structural alterationε in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that cDNA sequence. Ultimately, complete sequencing of geneε from several individuals is required to confirm the presence of a mutation and to distinguish mutations from polymorphisms. The present invention is further directed to inhibiting haemopoietic maturation factor in vivo to reduce and/or eliminate itε stimulatory effect by use of antisense technology. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide sequence to DNA or RNA. For example, the 5' coding portion of the polynucleotide sequence, which encodes for the polypeptide of the present invention, is used to design an antisense RNA oligonucleotide of from 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix - see Lee et al, Nucl. Acids Reε. , 6:3073 (1979); Cooney et al. Science, 241:456 (1988); and Dervan et al. Science, 251: 1360 (1991), thereby preventing tranεcription and the production of haemopoietic maturation factor. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of an mRNA molecule into the haemopoietic maturation factor (antisense - Okano, J. Neurochem.. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression. CRC Press, Boca Raton, FL (1988)). -21- SUBSmUTE SHEET (WLt 26) Alternatively, the oligonucleotideε described above can be delivered to cells by procedures in the art such that the anti-senεe RNA or DNA may be expressed in vivo to inhibit production of haemopoietic maturation factor in the manner described above. Antisense constructε to haemopoietic maturation factor, therefore, may stimulate cell growth which may be used for treating certain disorderε, for example, AIDS, since blocking haemopoietic maturation factor may control T-cell maturation. In this way, antisense constructε may also be used to treat anemia. The protein, itε fragmentε or other derivativeε, or analogε thereof, or cellε expressing them can be used as an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal, monoclonal, chimeric, single chain. Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of polyclonal antibodies. Antibodieε generated against the polypeptide corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptide into an animal or by administering the polypeptide to an animal, preferably a nonhuman. The antibody εo obtained will then bind the polypeptide itεelf. In thiε manner, even a sequence encoding only a fragment of the polypeptide can be used to generate antibodies binding the whole native polypeptide. Such antibodies can then be used to isolate the polypeptide from tisεue expreεεing that polypeptide. Moreover, a panel of εuch antibodieε, specific to a large number of polypeptides, can be uεed to identify and differentiate such tissue. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature. 256:495-497), the trioma technique, the human B-cell hybrido a technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Lisε, Inc., pp. 77- 96). Techniqueε described for the production of single chain antibodies (U.S. Patent 4,946,778) can be adapted to produce εingle chain antibodies to immunogenic polypeptide products of this invention. The antibodies can be used in methodε relating to the localization and activity of the protein εequenceε of the invention, e.g., for imaging theεe proteins, measuring levelε thereof in appropriate phyεiological εampleε and the like. Antibodies specific to the haemopoietic maturation factor may further be used as antagonists to inhibit the proper functioning of the polypeptide. In this manner, the antibodies may be used in therapy, for example, to stimulate cell growth to treat certain disorderε, for example, AIDS and anemia. Further, such antibodies can detect the presence or absence of haemopoietic maturation factor and, therefore, are indicators of disorders of the haemopoietic and neural system. These antibodieε may, therefore, be used as a diagnoεtic for disorders associated therewith. Prenatal diagnosis may also be particularly useful where the associated disorders are congenital. Alternatively, an antagonist to the polypeptide of the present invention may be employed which binds to the receptors to which the polypeptide of the present invention normally binds. The antagonist is similar to an inactive form of the polypeptide and may be generated in ways similar to the ways in which antibodies specific to haemopoietic maturation factor are generated. In this way the action of the haemopoietic maturation factor is prevented and the antagonist may be used therapeutically for stimulation of cell growth to treat certain disorders, for example AIDS. The antagonist may also be used diagnoεtically to detect the preεence or abεence of haemopoietic maturation factor. The preεent invention will now be further deεcribed with reference to the following exampleε; however, it iε to be underεtood that the present invention iε not limited to such exampleε. All partε or amountε, unleεε otherwise specified, are by weight. In order to facilitate understanding of the following exampleε, certain frequently occurring methodε and/or terms will be described. "Plasmidε" are deεignated by a lower caεe p preceded and/or followed by capital letters and/or numbers. The starting plasmidε herein are either commercially available, publicly available on an unrestricted baεiε, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan. "Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequenceε in the DNA. The variouε restriction enzymes uεed herein are commercially available and their reaction conditionε, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, typically 1 μg of plasmid or DNA fragment iε used with about 2 units of enzyme in about 20 μl of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, typically 5 to 50 μg of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37°C are ordinarily used, but may vary in accordance with the supplier's instructions. After digeεtion the reaction is electrophoreεed directly on a polyacrylamide gel to isolate the desired fragment. Size separation of the cleaved fragments iε performed uεing 8 percent polyacrylamide gel deεcribed by Goeddel, D. et al . , Nucleic Acidε Reε. , 8:4057 (1980). "Oligonucleotideε" refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phoεphate and thuε will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinaεe. A εynthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated. "Ligation" referε to the proceεε of forming phosphodiaster bonds between two double stranded nucleic acid fragments (Maniatis, T., et al.. Id. , p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase ("ligase") per 0.5 μg of approximately equimolar amounts of the DNA fragments to be ligated. Example 1 Expreεεion and purification of the haemopoietic maturation factor. The DNA sequence encoding for haemopoietic maturation factor (ATCC # 75514) is initially amplified uεing PCR oligonucleotide primerε corresponding to the 5' and 3' end of the DNA sequence to synthesize insertion fragments. The 5' oligonucleotide primer has the sequence GACTTCATGAAAAAGACAGATATCGCAATTGCAGTGGCAC- TGGCTGGTTTCGCTACCGTTGCGCAAGCTGCTTCTGACTCCCTGGTGGTGTGC contains a BspHI restriction enzyme site and the ompA -25- SUBSWTUTESHEET(RULE26) leader sequence followed by 21 nucleotides of haemopoietic * maturation factor coding sequence starting from the codon following the ethionine start codon; the 3' sequence GACTAGATCTACGAAAGAAAGACAACTTTTC contains complementary sequences to Bglll site, and the last 21 nucleotideε of haemopoietic maturation factor coding εequence. The reεtriction enzyme sites correspond to the restriction enzyme siteε on the bacterial expreεsion vector pQE-60 (Qiagen Inc., 9259 Eton Ave., Chatsworth, CA 91311). The plasmid vector encodes antibiotic resistance (Amp r ), a bacterial origin of replication (ori), an IPTG-regulatable promoter/operator (P/0), a ribosome binding εite (RBS), a 6-histidine tag (6-His) and reεtriction enzyme cloning sites. The pQE-60 vector was digested with Ncol and Bglll and the insertion fragmentε were then ligated into the pQE- 60 vector maintaining the reading frame initiated at the bacterial RBS. The ligation mixture waε then used to transform the E. coli strain ml5/rep4 (available from Qiagen under the trademark ml5/rep4). M15/rep4 contains multiple copies of the plasmid pREP4, which expresεeε the lad repressor and also confers kanamycin resistance (Kan r ). Transformantε are identified by their ability to grow on LB plateε containing both Amp and Kan. Clones containing the desired constructs were grown overnight (O/N) in liquid culture in either LB media supplemented with both Amp (100 μg/ml) and Kan (25 μg/ml). The 0/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cellε were grown to an optical density of 600 (O.D. 600 ) between 0.4 and 0.6. IPTG ("Isopropyl-B-D-thiogalacto pyranoside") was then added to a final concentration of ImM. IPTG induces by inactivating the lad repressor, clearing the P/O leading to increased gene expreεsion. Cells were grown an extra 3-4 hours. Cells were then harvested by centrifugation. The cell pellet was -26- SUBSTITUTE SHEET {RULE 26) solubilized in the chaotropic agent 6 Molar Guanidine HCL. - After clarification, solubilized haemopoietic maturation factor was purified from this solution by chromatography on a Nickel-Chelate column under conditions that allow for tight binding by proteins containing the 6-Hiε tag. (Hochuli, E. et al.. Genetic Engineering. Principle & Methods, 12:87-98 Plenum Preεε, New York (1990)). Haemopoietic maturation factor (95% pure) waε eluted from the column in 6 molar guanidine HCL pH 5.0 and for the purpoεe of renaturation adjusted to 3 molar guanidine HCL, lOOmM sodium phoεphate, 10 mmolar glutathione (reduced) and 2 mmolar gluthatione (oxidized). After incubation in thiε solution for 12 hours the protein was dialyzed to 50 mmolar sodium phosphate. Example 2 Expression of RNA encoding the haemopoietic maturation factor in different tisεueε. In order to analyze the expression pattern of the haemopoietic maturation factor RNA was isolated from different human tisεueε. After total RNA isolation poly A containing RNA was isolated by affinity chromatography on a poly T containing affinity resin. Poly A containing RNA (2 ug) was εeparated by denaturing gel chromatography and the poly A containing RNA tranεferred to a membrane by blotting RNA expression pattern waε determined by hybridizing the membrane bound poly A RNA to a radioactive probe spanning the haemopoietic maturation factor gene followed by auto radiography. Figure 4. Example 3 Haemopoietic maturation factor and inhibition of growth of HeLa cells. HeLa cells were plated at 10 5 (experiment #1) or 10 4 (experiment #2 and #3) per well in 24 well plates. Cells were grown in DMEM ("Dulbecco's Modified Eagle's Medium") medium (1 ml) containing 5% fetal calf serum penicillium 50 units/ml and streptomycin 50 ug/ml. Four wells were plated for each concentration of haemopoietic maturation factor per experiment. Haemopoietic maturation factor waε applied one day after plating. After 3 days cells were trypsinized and counted. Figure 5. The reεults indicate that haemopoietic maturation factor inhibits the growth of cancer cellε, εince Hela cellε are formε of cancer cellε. Numerouε modificationε and variationε of the preεent invention are possible in light of the above teachings and, therefore, within the scope of the appended claims, the invention may be practiced otherwiεe than aε particularly described. -28- SUBSTlTUTt SHEET (RU1.E 26) SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: KIRKNESS, ET AL. (ii) TITLE OF INVENTION: Haemopoietic Maturation Factor (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: CARELLA, BYRNE, BAIN, GILFILLAN, CECCHI, STEWART & OLSTEIN (B) STREET: 6 BECKER FARM ROAD (C) CITY: ROSELAND (D) STATE: NEW JERSEY (E) COUNTRY: USA (F) ZIP: 07068 (V) COMPUTER READABLE FORM: (A) MEDIUM TYPE: 3.5 INCH DISKETTE (B) COMPUTER: IBM PS/2 (C) OPERATING SYSTEM: MS-DOS (D) SOFTWARE: WORD PERFECT 5.1 (Vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: Concurrently (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 08/187,186 (B) FILING DATE: 25 JAN 1994 (Viii) ATTORNEY/AGENT INFORMATION: (A) NAME: FERRARO, GREGORY D. (B) REGISTRATION NUMBER: 36,134 (C) REFERENCE/DOCKET NUMBER: 325800-143 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 201-994-1700 (B) TELEFAX: 201-994-1744 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 600 BASE PAIRS (B) TYPE: NUCLEIC ACID (C) STRANDEDNESS: SINGLE (D) TOPOLOGY: LINEAR (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: AGACAGCGGA ACTAAGAAAA GAAGAGGCCT GTGGACAGAA CAATCATGTC TGACTCCCTT 60 GTGGTGTGCG AGGTAGACCC AGAGCTAACA GAAAAGCTGA GGAAATTCCG CTTCCGAAAA 120 GAGACAGACA ATGCAGCCAT CATAATGAAG GTGGACAAAG ACCGGCAGAT GGTGGTGCTG 180 GAGGAAGAAT TTCAGAACAT TTCCCCAGAG GAGCTCAAAA TGGAGTTGCC GGAGAGACAG 240 CCCAGGTTCG TGGTTTACAG CTACAAGTAC GTGCATGACG ATGGCCGAGT GTCCTACCCT 300 TTGTGTTTCA TCTTCTCCAG CCCTGTGGGC TGCAAGCCGG AACAACAGAT GATGTATGCA 360 GGGAGTAAAA ACAGGCTGGT GCAGACAGCA GAGCTCACAA AGGTGTTCGA AATCCGCACC 420 ACTGATGACC TCACTGAGGC CTGGCTCCAA GAAAAGTTGT CTTTCTTTCG TTGATCTCTG 480 GGCTGGGGAC TGAATTCCTG ATGTCTGAGT CCTCAAGGTG ACTGGGGACT TGGAACCCCT 540 AGGACCTGAA CAACCAAGAC TTTAAATAAA TTTTTAAATG CAAAAAAAAA AAAAAAAAAA 600 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 142 AMINO ACIDS (B) TYPE: AMINO ACID (C) STRANDEDNESS: (D) TOPOLOGY: LINEAR (ii) MOLECULE TYPE: PROTEIN (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Ser Asp Ser Leu Val Val Cys Glu Val Asp Pro Glu Leu Thr 5 10 15 Glu Lys Leu Arg Lys Phe Arg Phe Arg Lys Glu Thr Asp Asn Ala 20 25 30 Ala lie lie Met Lys Val Asp Lys Asp Arg Gin Met Val Val Leu 35 40 45 Glu Glu Glu Phe Gin Asn lie Ser Pro Glu Glu Leu Lys Met Glu 50 55 60 Leu Pro Glu Arg Gin Pro Arg Phe Val Val Tyr Ser Tyr Lys Tyr 65 70 75 Val His Asp Asp Gly Arg Val Ser Tyr Pro Leu Cys Phe lie Phe 80 85 90 Ser Ser Pro Val Gly Cys Lys Pro Glu Gin Gin Met Met Tyr Ala -31- SUBST1TUTESHEET(RULE26) 95 100 105 Gly Ser Lys Asn Arg Leu Val Gin Thr Ala Glu Leu Thr Lys Val 110 115 120 Phe Glu lie Arg Thr Thr Asp Asp Leu Thr Glu Ala Trp Leu Gin 125 130 135 Glu Lys Leu Ser Phe Phe Arg 140

Claims: WHAT IS CLAIMED IS: 1. An isolated DNA sequence comprising DNA encoding at least the same mature polypeptide as the DNA sequence of Figure 1 or any allelic variants thereof. 2. The isolated DNA of claim 1 wherein said DNA comprises the DNA of Figure 1 encoding for at least the mature polypeptide of Figure 1 and allelic variants thereof. 3. An isolated RNA sequence comprising RNA and allelic variants thereof corresponding to a DNA sequence of claim 1. 4. An isolated DNA sequence comprising a DNA sequence encoding at least a mature polypeptide identical to the mature polypeptide encoded by the DNA sequence contained in ATCC Deposit No. 75514 and allelic variants thereof. 5. The isolated DNA of claim 4 wherein said DNA comprises the DNA contained in ATCC Deposit No. 75514 which encodes the mature polypeptide. 6. An isolated RNA sequence comprising RNA corresponding to the DNA sequence of claim 4. 7. An expression vehicle comprising the DNA sequence of claim 1. 8. An expression vehicle comprising the DNA sequence of claim 4. 9. A polypeptide comprising an amino acid sequence encoded by the DNA sequence of claim 1 or an active fragment, derivative or functional analog thereof. 10. A polypeptide comprising an amino acid sequence encoded by the DNA sequence of claim 4 or an active fragment, derivative or functional analog thereof. 11. A polypeptide comprising an amino acid seguence encoded by the DNA sequence of claim 2 or an active fragment, derivative or functional analog thereof. 12. A polypeptide comprising an amino acid sequence encoded by the DNA sequence of claim 5 or an active fragment, derivative or functional analog thereof. 13. A process for producing a polypeptide comprising: expressing a polypeptide by use of the DNA of claim 1. 14. An antibody against the polypeptide of claim 9. 15. An antibody against the polypeptide of claim 10. 16. Cells engineered with DNA of claim 1. 17. A process for producing cells for expressing a polypeptide comprising: genetically engineering cells with the DNA of claim 1. 18. An antagonist against a polypeptide of claim 9. 19. A method for the treatment of a patient having need of a haemopoietic maturation factor comprising: administering to the patient a therapeutically effective amount of the polypeptide of claim 9. 20. A method for the treatment of a patient to inhibit a haemopoietic maturation factor comprising: administering to the patient a therapeutically effective amount of the antagonist of claim 18. 21. An isolated DNA sequence hybridizable to the DNA sequence of claim 1. 22. The method of Claim 19 wherein εaid therapeutically effective amount of εaid polypeptide iε adminiεtered to a patient by providing to the patient DNA encoding εaid polypeptide and expressing said polypeptide in vivo .

Inventors: ADAMS MARK D, KIRKNESS EWEN, OLSEN HENRIK, ROSEN CRAIG, ADAMS, MARK, D., KIRKNESS, EWEN, OLSEN, HENRIK, ROSEN, CRAIG
Applicants: HUMAN GENOME SCIENCES INC, HUMAN GENOME SCIENCES, INC.
Application year: 1994
Main classification: C07H17
IPC codes: A61K38, A61K39, A61K48, A61P7, A61P25, A61P35, A61P43, C07H21, C07K14, C07K16, C12N1, C12N5, C12N15, C12P21, C12Q1